1. Field of the Invention
This invention relates generally to a method of separating and purifying factor VIII procoagulant activity protein. More specifically, high purity factor VIII procoagulant activity protein is separated from von Willebrand Factor by a two step chromatographic adsorption and concentration technique from plasma or concentrate.
2. Description of the Prior Art
The isolation of the antihemophilic factor from blood plasma has been described in the literature. The precise structure of the antihemophilic factor, also referred to as factor VIII procoagulant activity protein (factor VIII), has not yet been identified, due in part to the unavailability of sufficient quantities of pure material with which to conduct further studies. The limited availability of pure material and its existence in a dilute state has also hindered its use in therapeutic applications.
Factor VIII procoagulant activity protein functions to correct the clotting defect in hemophilic plasma. It circulates in plasma complexed with the von Willebrand factor protein. The latter can alter the platelet function defect in von Willebrand's disease. That portion of the factor VIII von Willebrand factor complex having coagulant activity is referred to as factor VIII procoagulant activity protein, factor VIII-clotting activity or simply VIII:C (the designation of "VIII:C" will be used hereinafter to identify the portion of the factor VIII molecule with such clotting activity.) The other portion of the factor VIII von Willebrand factor complex having the ability to correct the platelet function defect in von Willebrand's disease is referred to as von Willebrand factor, factor VIII-related antigen, VIIIR:Ag, VIII:RP factor. (The description "VIII:RP" will be used hereinafter to identify the platelet correction function of the factor VIII molecule). Although yet unproven, there is evidence to support the conclusion that VIII:C exhibits properties and the behavior of a small molecule which is combined with VIII:RP as a non-covalent complex. There is also a basis for the contention that the properties associated with both VIII:C and VIII:RP may also be a single molecule which under appropriate conditions may be cleaved, yielding two fragments.
In view of the need for identifying the structures of the factor VIII/von Willebrand factor complex, VIII:C and VIII:RP and the important pharmaceutical value of the coagulant activity ascribable to VIII:C, numerous attempts have been made to purify factor VIII and to separate and concentrate VIII:C and VIII:RP. The techniques used are based generally on either immunoadsorption or ion exchange chromatography. Such techniques as heretofore used have had limited success due to the difficulty of desorbing the proteins from the charged ionic material in an undamaged condition or recovering same in suitable quantities.
One such method for separating VIII:C from VIII:RP utilizing immunoadsorbent chromatography has been reported by E. G. D. Tuddenham et al, "The Properties of Factor VIII Coagulant Activity Prepared by Immunoadsorbent Chromatography", JOURNAL OF LABORATORY CLINICAL MEDICINE, Vol. 93, p. 40 (1979). The reported method is a one-step separation of VIII:C from nearly all VIII:RP and from most other plasma proteins employing a chromatographic column packed with agarose beads to which polyclonal antisera to VIII:RP (anti-VIII:RP) are coupled. Factor VIII/von Willebrand factor containing plasma is passed through the column which adsorbs both VIII:C and VIII:RP. Other unwanted plasma proteins are removed from the column by washing with buffered saline solution and the desired VIII:C is obtained by subsequent elution with a calcium-ion gradient. Although it is stated to be an improvement in both purity and yield of VIII:C, when compared to the previously known methods, it is also stated that the resulting product also contains VIII:RP and other plasma proteins. Such contaminants may be attributable to the use of polyclonal antisera bound to the agarose beads. Since a majority of the immunoglobulins from which the antisera are constituted are not specific to VIII:RP, the effective number of sites where antibodies specific to VIII:RP may be bound to agarose is relatively small due to competition between the antisera for a finite number of bonding sites on the agarose.
Another method for separating VIII:C from VIII:RP and ristocetin co-factor by a chromatographic technique employing aminohexyl-substituted agarose has been described by D. E. G. Austen, "The Chromatographic Separation of Factor VIII on Aminohexyl Sepharose," BRITISH JOURNAL OF HAEMATOLOGY, Vol. 43, p. 669 (1979). The described method is stated to be an improved method for the component parts of both human and porcine factor VIII/von Willebrand factor. This method, however, also suffers from the fact that contaminants are present in the resulting product. In both the Tuddenham et al and Austen methods a contaminated product, which is more dilute than is normally desired, is formed.
Hence, it is clear that there still exists a need for an improved method for separating and purifying VIII:C from VIII:RP using plasma or concentrates. Therefore, it is an object of the present invention to satisfy such a need.